Inverse of Rensch’s Rule of Allometry for Sexual Size Dimorphism in Bufo andrewsi

Difference in body size between males and females(sexual size dimorphism:SSD)and its variation are a common phenomenon in animal kingdom.Rensch’s rule predicts that the degree of SSD variation increases with the enlarged mean body size when males are larger than females and decreases when females are larger than males.Here,whether the patterns of variations in SSD in the Andrew’s toad(Bufo andrewsi)follow Rensch’s rule was tested using unpublished data from 14 populations and published data from 17 populations.Results show the reduced major axis regression of log10(male size)on log10(female size)across all populations displayed a significant hyperallometric relationship,which was consistent with inverse Rensch’s rule(the degree of SSD increased with enlarged mean body size).SSD could also be explained by sexual age difference(SAD)due to a positive SSD–SAD relationship among all populations.The findings suggest that the occurrence of inverse Rensch’s rule in B.andrewsi is likely to be a result of fecundity selection on increased reproductive investments in larger females.

Bufo gargarizans:What’s in a Name?

The taxonomic status of Bufo species in East Asia,particularly those closely related to B.gargarizans,has been controversial for over half a century,and recent molecular studies further fueled the dispute.Whether B.andrewsi,B.bankorensis,B.minshanicus,B.sachalinensis,B.tibetanus,and B.tuberculatus constitute valid species or are geographic or ecological variants of B.gargarizans is at the centre of the discussion.Most of these forms possess morphological diagnostic traits,but those differences are minimal and often inconsistent.Molecular data produced many contradictory patterns.Lack of clear diagnostic characters,different usage or definition of various names,mis-identified specimens,limited amount of data or samples,and frequent inter-specific hybridization may all have contributed to the current controversies.Some contradiction may also result from differences between similarity-based clustering and gene tree-based branching patterns.Nevertheless,a dominant allopatric differentiation pattern within this species complex is apparent,and several regional population groups have been identified.Some of the groups resemble old names(e.g.B.minshanicus,B.tibetanus),while others are new.These groups may represent geographic or ecological variants,incipient species,or valid species,and the current data do not warrant a convincing taxonomic revision.To move forward,a single study with comprehensive sampling and genomic data is needed to establish a solid framework,which will define naturally existing population clusters/lineages,and detect their levels of divergence and approximate geographic boundaries.Morphological and ecological examination will follow to understand their biology,determine their taxonomic status,and identify consistent diagnostic characters if necessary.

New Karyological and Morphometric Data on Poorly Known Bufo surdus and Bufo luristanicus in Comparison with Data of Diploid Green Toads of the Bufo viridis Complex from South of Iran

Previous studies on the Bufo viridis complex, which is distributed broadly across Iran, are incomplete and restricted to a few regions or a few samples. In this paper a new detailed study on the B. viridis complex in southern of Iran （from West to East） is presented. The analysis of 18 morphometric characters with univariate and multivariate methods reveals significant differences between three members of the B. viridis complex namely B. variabilis, B. luristanicus, and B. surdus distributed in southern part of Iran. Our result help to resolve an old taxonomic problem about B. surdus subgroup （taxa closely related to B. surdus） confirming that B. luristanicus and B. surdus are distinct species. Moreover, for the first time we report and describe karyotype details of B. luristanicus and B. surdus which confirmed that they are diploid. Karyological studies demonstrate that all toads from three mentioned species have 2n = 22 chromosomes. These chromosomes are arranged into two groups. First group has six large chromosomes and the second group is composed of five small chromosomes. These chromosomes are metacentric or submetacentric. The number of submetacentric chromosomes is different in three mentioned species of B. viridis complex. Neither sexual heteromorphism, nor secondary constriction was observed in any pairs of chromosomes.

cDNA Cloning and Expression Analysis of Mest Gene in the Bufo gargarizans

The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5＇- and 3＇-RACE from the Chinese Large Toad （Bufo gargarizans）. The transcript is 1 325bp in length which contains a complete open reading frame （ORF） encoding a polypeptide of 326 amino acids （GenBank accession number： ABQ10905）. There is a typical 0./13 hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali （86%）, mammlian （70% - 80%）. RT-PCR （reverse transcriptase-polymerase chain reaction） analysis demonstrated that the Bufo gargarizans Mest （BgMest） gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.

Cloning the sterol carrier protein 2 genes of Japanese toad （Bufo japonicus formosus） and Chinese toad （Bufo gargarizans） and its tissue expression analysis

In this study,to clarify the bioactive polypeptides included in the skins and secretions of Bufo,we screened the Japanese toad(Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction(PCR),and obtained a transcript of 1 075 bp consisting of 1 37 bp 5′ untranslated region(UTR),515 bp 3′ UTR and a 423 bp open reading frame(ORF) encoding a polypeptide of 140 amino acid residues(GenBank accession number: KF359945).Homolog analysis showed a 70%–96% homology with sterol carrier protein-2(SCP-2) present in other animals,which is implicated in lipid metabolism of other organisms.The gene SCP-2 of Chinese toad(B.gargarizans) was cloned from a first strand cDNA of Bufo skin(GenBank accession number: KF381341) via PCR,whose encoding polypeptide has only one amino acid difference from that of Japanese toad.Tissue distribution analysis showed that SCP-2 expressed in all organs tested,though in the liver and spleen it manifested lower expression than in other organs.These findings might indicate SCP-2 being one of the active ingredients in toad skin.These findings may in turn have implications for further drug development from traditional Chinese medicine sources.

Changes of Spontaneous Electrical Activities of Olfactory Bulb in Bufo gargarizans during Postembryonic Development

Objective] The aim of this study was to investigate the electrophysiological characteristics of spontaneous electrical activities of olfactory bulb in Bufo gargarizans during postembryonic development. [Method] Microelectrode technique was used to record extracellular spontaneous electrical activities of olfactory bulb in B. gar-garizans. [Result] Along with the development of olfactory bulb, the discharge types increased gradual y. Single spontaneous firing enhanced gradual y and it reached the maximum in the metamorphosis phase. The intensity of consecutive single sponta-neous firing changed slightly at the middle of post-embryonic development, but it was stil higher than in the early phase and the adult phase. Consecutive burst spontaneous firing frequency went down over time. [Conclusion] Along with the de-velopment of olfactory bulb, the neuronal excitability increases step by step and the neuronal electrical activities diversify gradualy.

草甘膦胁迫对中华大蟾蜍(Bufo gargarizans)神经冲动产生和传导的影响

应用电生理方法研究了除草剂草甘膦对中华大蟾蜍（Bufo gargarizans Cantor）坐骨神经干冲动产生和传导的影响。用不同浓度的草甘膦溶液对中华大蟾蜍进行胁迫处理,草甘膦有效成分经由皮肤进入蟾蜍体内而作用于神经系统,利用生物信号采集处理系统测定草甘膦胁迫下中华大蟾蜍离体坐骨神经干的应激反应时间、动作电位幅度和冲动传导速度,结果表明：随着草甘膦溶液浓度的升高,中华大蟾蜍坐骨神经干接受刺激后产生冲动所需的时间逐渐延长,动作电位峰值降低,神经冲动传导速度亦逐渐减慢。草甘膦施用后,中华大蟾蜍7d内的平均应激反应时间与草甘膦浓度呈正相关,而动作电位幅度及传导速度均与草甘膦浓度呈负相关。草甘膦溶液浓度达到推荐农田使用浓度1.64~2.87ml/L时,各处理组蟾蜍的应激反应时间、动作电位幅度和冲动传导速度均与对照组差异极显著（P〈0.01）。同时,随着试验处理时间的延长,中华大蟾蜍神经干对刺激的反应变得更为迟钝,神经冲动的传导速度也进一步减慢。回归分析可知,中华大蟾蜍坐骨神经干的应激反应时间与草甘膦施用后天数呈正相关,而神经传导速度与药后天数呈负相关。由此可以说明,草甘膦胁迫条件下,中华大蟾蜍神经细胞对刺激反应的灵敏性降低，动作电位的产生及传导受到一定程度的抑制和阻碍。

Advertisement Call Variability in the Black-spined Toad Bufo melanostictus (Anura: Bufonidae) during the Breeding Season in Lishui, Zhejiang, China

Advertisement calls play an important role in influencing female mate choice and are regarded as one of the key characters responsible for reproductive isolation and speciation events in frogs and toads. The black-spined toad, Bufo melanostictus, is one of the most common amphibians in Southeast Asia and is frequently observed in human habitations throughout its distributional range. The advertisement calls of the toad have not been well described. Here, we report the advertisement calls of this species from Lishui, Zhejiang, China during its breeding season. The advertisement calls of B. melanostictus are composed of single notes with a single harmonic frequency emitted sporadically. The call parameters recorded in this study were highly variable across recording times and with different body sizes. Dominant frequency was negatively correlated with body size. Comparing the calls of the B. melanostictus population from Lishui with the populations from five other localities(Bangkok, Thailand; Coorg, India; Burma, Myanmar; Bali, Indonesia; Halimun-Salak National Park, Indonesia), we found that call structure was similar among five different populations with no harmonics, while the Halimun-Salak National Park population contained two clear harmonics. Dominant frequency was similar between Bali(1.56 kHz) and Coorg(1.6 kHz) populations, while it was highly variable among other populations. Besides call duration, call interval and pulse/call rate may also be significantly different among the comparing populations. Pooling the available data, we observed some similarities and differences in call parameters among the six populations.

Postembryonic Development of Electrophysiological Characteristic of Primordial Hippocampus in Bufo bufo gargarizans

[Objective] The aim was to probe into developing changes of spontaneous electrical activities of primordial hippocampus in Bufo bufo gargarizans in postembryonic phase. [Method] Microelectrode technique was used to record extracellularly spontaneous electrical activities of neurons in primordial hippocampus of B.bufo gargarizans in postembryonic phase. [Result] Single, consecutive single, burst, consecutive burst and anomalous spontaneous firing were recorded in primordial hippocampus, in which four kinds of spontaneous firings were recorded in forepart of postembryonic development except the anomalous spontaneous firing, and the burst spontaneous firing and consecutive burst spontaneous firing were predominant. The five kinds of spontaneous firings mentioned were recorded in anaphase of postembryonic development and the single and burst spontaneous firing were predominant.Following with the development of primordial hippocampus, the amplitudes of action potential of single firing and consecutive firing enhanced gradually, and it reaches the maximum in the metamorphosis phase; the differences of the frequencies of consecutive single firing and consecutive burst firing were not significant. [Conclusion] Following with the development of primordial hippocampus, the neuronal electrical activities showed diversification, the developmental degree of the primordial hippocampus in the metamorphosis phase was close to the subadult and adult.

Spontaneous firing in olfactory bulb neurons of Bufo bufo gargarizans in and after hibernation

Microelectrode technique was used to record the spontaneous electrical activities of the neurons in olfactory bulb of the Bufo bufo gargarizans, both in hibernation and after hibemation. This study investigated the electrophysiological characteristics of amphibian olfactory bulb in the period of hibernation and after hibernation and its effects on the start of hibernation and spontaneous awakening. The research showed four forms of spontaneous firings： single spontaneous firing, burst spontaneous firing, irregular spontaneous firing and consecutive single spontaneous firing. The single spontaneous firing includes slow depolarized spontaneous firing and fast depolarized spontaneous firing, and the slow depolarized spontaneous firing occurs only during the hibernation period. In hibernation, the low amplitude and low frequency firing with a longer duration may be relevant to maintaining the tonicity of the central nervous system in toads that are in hibernation, and this kind of firing may also provide an excited basis for their arousal from hibernation. After hibernation, the amplitude and frequency of firing increase, but the firing duration gets shorter. This form of short-term firing, which may be a phenomenon of sensory neurons fast adapting, is one of the neuronal mechanisms for the arousal of hibernating animals.

绿蟾蜍(Bufo viridis Laurenti)的早期胚胎发育和分期

本文报道了新疆常见的无尾两栖类——绿蟾蜍(Bufo viridis Laurenti)的胚胎发育和分期。根据胚胎的外部形态变化特征,绿蟾蜍的早期胚胎发育可划分为25个时期;在20±1℃条件下,从人工授精开始到蝌蚪的鳃盖完全闭合需186.1h。绿蟾蜍的早期发育过程同国内已研究过的其他蟾蜍相似。

Sexual Dimorphism in the Hindlimb Muscles of the Asiatic Toad(Bufo gargarizans)in Relation to Male Reproductive Success

In many anurans, the forelimb muscles of males are used to grasp females and are often heavier than those of females despite the larger female body size. Such sexual dimorphism in forelimb musculature is thought to result from sexual selection. In addition, the hindlimbs of frogs and toads play an important role in the reproductive process as amplectant males can expel rivals with robust hindlimbs through kicking. In this study, the sexual dimorphism in dry mass for six hindlimb muscles of the Asiatic toad （Bufo gargarizans） was investigated. The results showed that, when controlled for body size, the hindlimb muscle mass of males significantly exceeded that of females for every muscle. The hindlimb muscle mass of amplectant males was also significantly larger than that of non-amplectant males. These results suggested that if strong hindlimb muscles could improve mating success of males, sexual selection would promote the evolution of dimorphism in this character.

Anticancer Peptide from Chinese Toad （Bufo Bufo Gargarizans） Skin Enhanced Sensitivity to 5-Fu in Hepatocarcinoma cells （HepG2）

OBJECTIVE To investigate the antiproliferative and apoptogenic activities of peptide extracted from the Chinese toad （Bufo bufo gargarizans） skin （TSP） and its effects on hepatocarcinoma cell line. METHODS M1-F assay was used to detect the effects of TSP （50 lig/mL and 5 ug/mL） on the proliferation and viability of Hepatocarcinoma cell line （HepG2） and liver cell line （L-02）; Flow cytometry was used in DNA content analysis to determine the cell distribution in different phases of cell cycle; Annexin V-FITC/PI stained fluorescence-activated cell sorter （FACS） and transmission electron microscope （TEM） were used to detect the apoptosis of the treated cells. RESULTS TSP could not suppress the proliferation and viability of normal liver L-02 cells, but strongly inhibited the proliferation and viability of HepG2 cells; TSP （50 μg/mL） primarily arrested the HepG2 cells at G1 phase of the cell cycle; TSP （50μg/mL） induced apoptosis in HepG2 cells and enhanced the effects of 5-Fu. CONCLUSION TSP has potent antineoplastic activity against human hepatocarcinoma cells with little toxicity to normal liver cells and can enhance the effects of 5-Fu.

A new linearly-combined bi-exponential model for kinetic analysis of the isometric relaxation process of Bufo gastrocnemius under electric stimulation in vitro

There was a slow-relaxing tail of skeletal muscles in vitro upon the inhibition of Ca2+-pump by cyclopiazonic acid (CPA). Herein, a new linearly-combined bi-exponential model to resolve this slow-relaxing tail from the fast-relaxing phase was investigated for kinetic analysis of the isometric relaxation process of Bufo gastrocnemius in vitro, in comparison to the single exponential model and the classical bi-exponential model. During repetitive stimulations at a 2-s interval by square pulses of a 2-ms duration at 12 V direct currency (DC), the isometric tension of Bufo gastrocnemius was recorded at 100 Hz. The relaxation curve with tensions falling from 90% of the peak to the 15th datum before next stimulation was analyzed by three exponential models using a program in MATLAB 6.5. Both the goodness of fit and the distribution of the residuals for the best fitting sup- ported the comparable validity of this new bi-exponential model for kinetic analysis of the relaxation process of the control muscles. After CPA treatment, however, this new bi-exponential model showed an obvious statistical superiority for kinetic analysis of the muscle relaxation process, and it gave the estimated rest tension consistent to that by experimentation, whereas both the classical bi-exponential model and the single exponential model gave biased rest tensions. Moreover, after the treatment of muscles by CPA, both the single exponential model and the classical bi-exponential model yielded lowered relaxation rates, nevertheless, this new bi-exponential model had relaxation rates of negligible changes except much higher rest tensions. These results suggest that this novel linearly-combined bi-exponential model is desirable for kinetic analysis of the relaxation process of muscles with altered Ca2+-pumping activity.

中华大蟾蜍(Bufo bufo gargarizans)早期胚胎发育的初步观察

中华大蟾蜍早期胚胎发育分为 2 5个时期 ,在常温下 (1 5 - 1 8℃ )全过程历时 393 .37小时。同时对早期胚胎发育的特点进行了讨论 。

中华大蟾蜍(Bufo bufo gargarizans Contor)早期胚胎发育中RNA的合成

以~3H-尿嘧啶核苷作为RNA合成的标记前身物,用放射自显术研究了中华大蟾蜍早期胚胎发育中RNA合成的速率及其与发育之间的关系。结果表明:从受精卵开始至原肠胚期,RNA的合成活性逐渐增强,神经胚期略有下降;RNA合成速率有明显的动—植物极梯度;在诱导与被诱导部位,RNA合成速率明显高于其它部位.

Hematological Parameters in Bufo arenarum Injected With Sublethal Dose of Lead Acetate

Lead has been recognized as a high risk toxic for most organisms including human. The effects of Pb in non-mammalian vertebrates are oprly known, particularly in anuran amphibians. The purpose of this study was to determine the effect of this metal on some hematological parameters of adult Bufo arenarum. It was found that all parameters remained unaltered within normal ranges, with the exception of reticulocyte counts which was significantly increased compared to the in controls (3. 7% vs. 0. 2 % ). It is suggested that the selective change found in reticulocyte count might be considered as an early response of a biomarker to sublethal exposition of Bufo arenarum to lead.

中华蟾蜍指名亚种(Bufo gargarizan gargarizan)早期胚胎发育

2006年1—2月份对中华蟾蜍指名亚种的早期胚胎发育和产卵行为进行观察.结果表明,蟾蜍整个产卵时间为6.18±0.73 h(N=4),其中雌性产卵8.00±1.20次,雄性射精9.25±1.31次.产卵中,雌性产卵的时间间隔为0.75±0.06 h(N=39),抱对的蟾蜍要游到水面上呼吸,两次呼吸间隔为1.39±0.23 h(N=11).雌蟾蜍最后一次产卵到雌雄分开所需时间为0.96±0.19 h(N=7).从受精卵期至鳃盖完成期,胚胎发育可分为23个时期,在6℃～10℃的水温条件下,历时1111.23±21.64 h(N=7).

Keratin expression during early embryonic development of Bufo bufo gargarizans

Three anti-keratin MAbs were used to identifykeratins expressed in early embryos of Bufo bufogargarizans.MAb AF5 recognized three polypeptides ofkeratin in oocytes,fertilized eggs,up to neurula withMr of 68,65 and 60Kd respectively.At tailbud stage,three other keratins(62,58 and 54Kd)began to expressand could be detected by AF5.MAbs D10 and K12 gavedifferent results,both of them could identify four keratin-like molecules with unusual molecular weights(Mr 98,95,30 and 27 Kd).Moreover,D10 could also detect a 54 Kdkeratin in neurula and tailbud stage embryos,while K12could reveal,beside 54Kd keratin,other four more kera-tins(68,65,62 and 60 Kd).The possible interpretation ofthese results and their implications are discussed.

Phylogenetic,Demographic and Dating Analyses of Bufo gargarizans Populations from the Zhoushan Archipelago and China's Mainland

Relatively little is known about the relationship between Bufo gargarizans populations from Zhoushan Archipelago and nearby continental regions on the Pacific coast of eastern China.In this paper,155 new specimens of B.gargarizans from Zhoushan Archipelago and adjacent continents and 71 published specimens of B.gargarizans from China's Mainland were studied.Phylogeographical and dating analyses of B.gargarizans were performed using mitochondrial DNA sequencing with a length of 1436 bp.A mt DNA tree that indicated seven major clades was obtained.The earliest split in the mt DNA tree corresponding to the divergence of populations from the western highland region occurred approximately 4.0 million years ago（mya）.A subsequent clade occurred about 3.4 mya,with cladogenesis continuing toward the end of the Pleistocene.The continental clades were distributed in the western,central and northeastern regions of China.Zhoushan Archipelago clades consisted of two largely geographically overlapping subclades with the mt DNA divergence time of 0.73 mya.These results indicated there was extensive dispersal after vicariance.The B.gargarizans populations on Zhoushan Archipelago most probably originated from populations in nearby eastern continental regions of China.It was concluded that geological uplifting during the Pliocene and several sea-level changes in Pleistocene might have influenced the divergence and population demographical history of this species.